US10139390B2 - Analysis device - Google Patents

Analysis device Download PDF

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US10139390B2
US10139390B2 US14/898,298 US201414898298A US10139390B2 US 10139390 B2 US10139390 B2 US 10139390B2 US 201414898298 A US201414898298 A US 201414898298A US 10139390 B2 US10139390 B2 US 10139390B2
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nanopore
temperature
dna
dna molecule
film
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US20160153960A1 (en
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Hirokazu Kato
Tomohiro Shoji
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Hitachi High Tech Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44791—Microapparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/60—Detection means characterised by use of a special device
    • C12Q2565/631—Detection means characterised by use of a special device being a biochannel or pore

Definitions

  • the present invention relates to an analysis device. More specifically, the present invention relates to a method for decoding a base sequence of a nucleic acid such as DNA or RNA and a nucleic acid sequence analysis device.
  • Nanopore technology has been performed popularly. “Advanced Sequencing Technology awards” which started to be granted by NHGRI (National Human Genome Research Institute, U.S.A.) in 2004 by using decoding the human genome in 2003 as a meter, was established in order to recommend technological development of a next-generation sequencer. The largest investment of these grants is the nanopore technology. The nanopore technology occupies 29 of 60 projects to which funds were granted from 2004 to 2010.
  • the nanopore includes bionanopore and solid nanopore.
  • the bionanopore treats protein and a lipid bilayer which are biomolecules. These substances are easily denatured disadvantageously.
  • One of big problems of the solid nanopore technology is that a passing speed of a DNA molecule passing through the nanopore is too high.
  • NPL 2 As a method for reducing the passing speed of DNA, in NPL 2, a method for adjusting the temperature, pH, viscosity, or the like of a solution, is examined.
  • Fologea et al. have succeeded in reducing the passing speed of a DNA molecule from 0.3 m/s to 1 mm/s by optimizing these conditions.
  • 1 mm/s is higher than 0.3 ⁇ m/s as an ideal speed by about four orders of magnitude, and it is necessary to further reduce the passing speed.
  • a signal of a blockage current used for detecting a DNA base is also reduced disadvantageously with the increase of the passing speed.
  • PTL 1 proposes a nanopore film having a film structure in which a plurality of conductive films and insulating films are stacked on each other. It is described that the passing speed of a DNA molecule passing through a nanopore can be controlled by using this nanopore film.
  • a cylindrical piezoelectric element is embedded in a nanopore opening. The piezoelectric element is in contact with the conductive film to be energized. The piezoelectric element can be extended or compressed by a nanometer scale according to a voltage applied thereto.
  • a DNA molecule in a solution is guided to the nanopore opening by an electrode disposed in a flow cell, invades the nanopore opening, and starts to pass through the nanopore.
  • the time when the DNA molecule starts to pass through the nanopore can be specified by detecting decrease in a blockage current.
  • a voltage is applied to the piezoelectric element embedded in the nanopore opening, and the piezoelectric element is expanded.
  • the DNA molecule in the nanopore can be thereby captured. It is possible to control the passing speed of the DNA molecule in the nanopore by controlling a potential with respect to the piezoelectric element in a pulse while an external electric field is applied to the DNA molecule. Alternatively, it is described that each base of the DNA molecule can go unidirectionally in the nanopore.
  • PTL 2 proposes a nanopore structure in which a gate electrode film is sandwiched by insulating films and the outside thereof is further sandwiched by a source electrode film and a drain electrode film.
  • This patent literature is also for controlling a passing speed of a DNA molecule in a nanopore. It is possible to control an orientation of ions in a solution including an electrolyte in the nanopore by further applying a gate voltage while a constant voltage is applied between the source and drain electrode films in advance. For example, it is possible to form a (negative, positive, negative) ion layer in the nanopore while the gate voltage is applied. On the other hand, it is possible to form a (negative, negative, negative) ion layer in the nanopore while the gate voltage is not applied.
  • a nanopore film is produced using an insulating film.
  • An organic molecule is modified to a nanopore opening in contact with DNA.
  • a weak and transitional bond (for example, hydrogen bond) is formed between this organic molecule and the DNA molecule. This bond is stronger than thermal fluctuation of the DNA molecule, and therefore the DNA molecule can be captured in the nanopore. It is described that a passing speed of DNA can be controlled by applying a voltage to the DNA molecule in a pulse in vertical upper and lower directions of the nanopore film.
  • a value of the electric conductivity of the peak value was the same as a value obtained by the monomer. It was also possible to measure the change of the electric conductivity for each base of the seven base RNA. It was found that the seven base sequence could be determined by arranging the above-described measurement sequences in a large amount like a shotgun sequence and performing statistical processing.
  • This model includes a ratchet having asymmetric teeth, an impeller, and a clasp on the wall.
  • This impeller is in a gas at a temperature T 1 . Therefore, a gas molecule collides with the impeller, and the impeller receives a random force clockwise or counterclockwise.
  • the temperature of the clasp is represented by T 2 .
  • a spring is attached to the clasp.
  • a single stranded DNA molecule is a polymer obtained by polymerizing molecules dATP, dCTP, dGTP, and dTTP which are asymmetric and have very similar structures. Therefore, the single strand DNA molecule has a spiral shape, and an asymmetric shape and a potential for each base on an axis thereof.
  • an elastic rod model which assumes symmetry of a molecule has been employed for rigidity of the DNA molecule.
  • experimental results in which the rigidity of the DNA molecule cannot be explained with this model have been submitted recently.
  • NPL 5 describes the rigidity of the DNA molecule with an asymmetric elastic rod model which assumes asymmetry of the DNA molecule.
  • Graphene As a material of the nanopore, graphene has attracted attention recently. Graphene is obtained by extending a hexagonal frame formed by carbon into a sheet-shape. Graphene is obtained by extracting one atomic surface of a graphite crystal. At present, measurement of a blockage current is employed most often in reading a base of DNA in the nanopore. When DNA passes through a nanopore, an effective area of the nanopore through which ions can pass changes. Therefore, a current flowing in upper and lower spaces of the nanopore also changes. This changing current is referred to as the blockage current. This blockage current is measured, and discrimination of four bases included in DNA is performed.
  • the thermal conductivity of graphene is highest in the currently known substances, and is 5000 [W/m/K]. On the other hand, the thermal conductivity of water is 0.6. There is a difference of about four orders of magnitude as the order of thermal conductivity.
  • the Young's modulus of graphene is highest in the currently known substances, and is 1500 [GPa].
  • An object is to improve accuracy of reading a DNA base sequence by lowering a passing speed of a DNA molecule in a nanopore (micropore in the order of nanometer).
  • the DNA passing through the nanopore is moved unidirectionally, and a passing speed in the nanopore is controlled.
  • the DNA molecule is a polymer obtained by polymerizing four kinds of nucleotides having very similar structures. Therefore, this structure has a periodic and asymmetric potential.
  • a temperature difference is introduced between the DNA molecule having an asymmetric potential and a nanopore substrate through which the DNA molecule passes.
  • a pawl in a molecular level is introduced into a nanopore opening. This pawl presses the DNA molecule with a spring, and holds the DNA molecule in the nanopore pore.
  • the DNA molecule in a solution holds the temperature in the liquid (temperature T 1 ), and the nanopore substrate is cooled to the temperature T 2 .
  • T 1 >T 2 .
  • the passing speed of a DNA molecule in a nanopore can be controlled and can be lowered. This can improve accuracy of analyzing a base sequence.
  • a double stranded DNA is unwound into a single stranded DNA according to the temperature, and the single stranded DNA can be measured selectively.
  • the polarity of the DNA molecule is selected, and the DNA molecule can be measured. It is possible to drive the same DNA molecule captured in the nanopore reversibly and unidirectionally, and to perform a sequence analysis in the same DNA molecule multiple times. Therefore, the accuracy of analyzing a base sequence can be improved.
  • the DNA molecule is driven discontinuously. Therefore, detection can be performed at a high S/N by time-averaging electric signals. An electric circuit disposed in the nanopore can be cooled. Therefore, detection accuracy can be improved.
  • FIGS. 1 a and 1 b are diagrams illustrating a device for extracting unidirectional motion by introducing a temperature difference between molecules in Example 1.
  • FIG. 2 is a diagram illustrating a device for analyzing a base sequence of a DNA single strand by introducing a temperature difference between molecules in Example 2.
  • FIGS. 3 a to 3 f are diagrams illustrating a method for preparing a single stranded DNA from a double stranded DNA and selecting a polarity of the DNA molecule passing through a nanopore in Example 3.
  • FIG. 4 is a diagram illustrating a method for analyzing a DNA base sequence by introducing a temperature difference between molecules and detecting a blockage current in Example 4.
  • FIG. 5 is a diagram illustrating a method for analyzing a DNA base sequence by introducing a temperature difference between molecules and detecting a tunneling current in Example 5.
  • FIG. 6 is a diagram illustrating a method for analyzing a DNA base sequence by introducing a temperature difference between molecules and including a spring function in a nanopore film in Example 6.
  • FIG. 7 is a diagram illustrating a method for controlling a speed of a DNA molecule passing through a nanopore by making an external electric field compete with a driving force caused by a temperature difference between molecules in Example 7.
  • FIG. 8 is a diagram illustrating a device for analyzing a base sequence of a DNA single strand by inverting a temperature difference between molecules at a high speed in Example 8.
  • FIGS. 9 a to 9 c are diagrams illustrating a method for analyzing a base sequence of a DNA single strand by inverting a temperature difference between molecules at a high speed in Example 9.
  • FIG. 10 is a diagram illustrating a flowchart of a method for analyzing a base sequence by introducing a temperature difference between molecules in Example 10.
  • FIGS. 1 a and 1 b a thermal ratchet device which is a method for moving a substance unidirectionally in a micro region, will be described with FIGS. 1 a and 1 b .
  • This device includes a ratchet 006 having asymmetric teeth, an impeller 002 , and a clasp 005 held on a wall 004 .
  • the ratchet 006 and the impeller 002 are connected to and fixed by an axis 003 .
  • This impeller 002 is in a box 001 including a gas at a temperature T 1 . Therefore, a gas molecule at the temperature T 1 collides with the impeller 002 , and the impeller 002 receives a random force clockwise or counterclockwise from the gas molecule.
  • the temperature of the wall 004 and the clasp 005 is represented by T 2 .
  • a spring 009 is attached to the clasp 005 .
  • Thermal motion of the linear ratchet 013 in the left and right directions moves a clasp 012 upward.
  • the ratchet 013 receives a horizontal kinetic energy and pushes the clasp 012 , the clasp 012 retracts and the ratchet 013 moves to the right side.
  • a probability at which the ratchet 012 receives energy is represented by [Mathematical Formula 1].
  • T 1 represents the temperature of the ratchet 013 .
  • T 2 The temperature of the clasp 012 is represented by T 2 .
  • a probability at which the clasp 012 retracts by itself is represented by [Mathematical Formula 2].
  • the ratchet 013 can move unidirectionally as represented by [Mathematical Formula 3].
  • a thermostatic bath 520 heats an aluminum plate 514 with a Peltier element 515 , and controls the temperature in the thermostatic bath 520 to T 1 by heating the air in the thermostatic bath 520 .
  • Heating control by the Peltier element 515 is performed by feedback-controlling a temperature value from a temperature measuring resistor 518 disposed in the aluminum plate 514 . More specifically, precise temperature control is performed by PID control.
  • PID control As concrete specifications of the thermostatic bath 520 , a region of adjusting the temperature is from 0 to 100° C., an allowable temperature difference is ⁇ 0.5° C., and temperature stability is SD ⁇ 0.06° C. (10 minutes).
  • a thermal protector 532 is disposed in the thermostatic bath 520 . When temperature runaway of 105° C. or higher occurs, supply of a voltage to the Peltier element 515 is cut off, and heating is stopped.
  • the Peltier element 515 When the Peltier element 515 is driven, heat transfer occurs between the surface and the back surface of the Peltier element 515 due to a Seebeck effect, and a temperature difference occurs.
  • a driving efficiency of the Peltier element 515 is lowered. Therefore, a fin 516 and a fan 517 are disposed on a Peltier surface in contact with the air outside in order to reduce the temperature difference.
  • a fan in bath 519 is disposed in order to make the temperature distribution T 1 of the air in the thermostatic bath 520 uniform.
  • thermostatic bath 520 This makes it possible to circulate the air heated by the aluminum plate 514 in the thermostatic bath 520 and to make the temperature in bath T 1 uniform.
  • the thermostatic bath 520 is covered with a heat insulating material 523 in order to avoid change in the temperature due to inflow and outflow of heat from a surrounding environment.
  • a flow cell 531 is disposed in the thermostatic bath 520 .
  • a solution including a DNA molecule 502 is injected into the flow cell 531 via a septum, and is equilibrated at the temperature T 1 in the thermostatic bath 520 .
  • a nanopore film 503 constituting a nanopore is stretched horizontally in the flow cell 531 .
  • the nanopore film 503 includes contact portions at both ends of the flow cell 531 outside, and these contact portions can be brought into contact with heat blocks 504 and 505 .
  • the heat blocks 504 and 505 can be cooled to the same temperature T 2 by driven Peltier elements 506 and 507 , respectively.
  • Temperature measuring resistors 512 and 513 which are temperature sensors are embedded in the heat blocks 504 and 505 , respectively.
  • Temperature control is performed by PID control.
  • the heat blocks 504 and 505 are equipped with heat insulating materials 521 and 522 , respectively, in order to prevent heat transfer by direct contact between the temperature T 2 of the heat blocks 504 and 505 and the temperature T 1 in the thermostatic bath 520 .
  • Fins 508 and 509 and fans 510 and 511 are attached to the Peltier elements 506 and 507 , respectively, in order to exhaust heat generated in the Peltier elements 506 and 507 .
  • the nanopore film 503 includes one or more nanopores 501 .
  • the DNA molecule 502 in the flow cell 531 is in a form of a double strand when a sample is injected, but is heated to the temperature in bath T 1 and is unwound into single strands.
  • the DNA molecule unwound into single strands is guided to a nanopore 501 opening by an external electrode disposed in the flow cell 531 .
  • the temperature of each of a solution including the DNA molecule and the DNA molecule 502 is T 1 , and is controlled stably.
  • the nanopore film 531 is cooled to the temperature T 2 by the Peltier elements 506 and 507 . This makes it possible to introduce a temperature difference between the DNA molecule 502 and the nanopore opening, and to realize the circumstances described in Example 1.
  • the DNA molecule 502 can be driven unidirectionally.
  • a driving speed of the DNA molecule 502 represented by [Mathematical Formula 3] to any speed.
  • the temperature range of T 1 is from 30 to 100° C., more specifically from 60 to 95° C., still more specifically 94° C.
  • the temperature range of T 2 is from 0 to 30° C., more specifically from 2 to 20° C., still more specifically 4° C.
  • the driving speed of the DNA molecule 502 is from 0.03 to 3 ⁇ m/s, more specifically 0.3 ⁇ m/s.
  • a conventional DNA sequencer requires an expensive optical system such as a CCD camera or an object lens, a driving unit using a motor, and an enzyme and a fluorescence reagent for performing a base extension reaction.
  • the analysis device reported in the present Example does not require the optical system or the driving unit.
  • the base extension is performed with an enzyme in a usual device.
  • the role is played by the temperature difference introduced between the molecules and asymmetry of the DNA molecule, and therefore the enzyme is not required. It is not necessary to exchange solutions, and therefore the device can be compact. Therefore, it is possible to provide a DNA sequencer which is very simple, inexpensive, robust, and compact.
  • DNA the sequence of which is to be analyzed is extracted from blood, urine, saliva, biopsy, a cultivation cell, a tissue section, or the like.
  • the DNA extracted and purified from these biological materials is not in a form of a single strand but in a form of a double strand. This is because complementary strands are bonded to each other in the DNA by forming a hydrogen bond, and the DNA turns into a form of a double strand and thereby minimizes free energy to be stabilized.
  • double strand single stranded DNA holding protein synthesis information is referred to as a sense strand, and the other single stranded DNA is referred to as an antisense strand.
  • a double stranded DNA molecule 601 prepared by a usual method is floating in a solution.
  • the solution in the thermostatic bath described in Example 2 is heated by temperature controlling by a Peltier element.
  • the temperature of the solution including the double stranded DNA molecule 601 is thereby raised to a temperature Tm at which the double stranded DNA molecule 601 is unwound into single strands, and the double stranded DNA molecule 601 is separated into a sense DNA molecule 602 and an antisense DNA molecule 603 which are complementary single strands.
  • Tm is referred to as melting temperature, and a temperature of 90 to 95° C.
  • the sense DNA molecule 602 is a polymer obtained by polymerizing four kinds of nucleotides.
  • the chemical structures of the four kinds of nucleotides are very similar to each other.
  • a DNA strand is formed by regularly stacking these nucleotides spirally.
  • This DNA strand is characterized in that the DNA strand has an asymmetric form periodically because nucleotide molecules having almost the same structure are stacked.
  • This asymmetric structure is not limited to a physical three-dimensional form, but may be a profile of an electrical interaction or a chemical interaction between molecules.
  • the sense DNA molecule 602 having a saw shape and an asymmetric and periodic form performs electrophoresis due to an electric field applied to a solution, and is guided to a nanopore 605 .
  • the electrophoresis can be performed by applying a voltage to an external electrode disposed in each of upper and lower regions separated by a nanopore film 604 in a flow cell. In this state, the temperature of the nanopore 604 is not lowered and is the same temperature T 1 as the solution. A spring mechanism is present near the nanopore 605 . Therefore, the sense DNA molecule 602 cannot invade the nanopore 605 , and performs a Brownian motion near the nanopore 605 due to an electric field.
  • the temperature of the nanopore film 604 is lowered to T 2 for the first time.
  • This cooling is performed by a Peltier element connected to the outside.
  • the temperature of the sense DNA molecule 602 is the same as the temperature T 1 to which the solution in the flow cell has been heated. This makes it possible to introduce a temperature difference T 1 ⁇ T 2 between the sense DNA molecule 602 and the nanopore film 604 .
  • the sense DNA molecule 602 has a saw shape having a periodic and asymmetric structure. Therefore, the sense DNA molecule 602 is driven downward by the introduced temperature difference T 1 ⁇ T 2 . This driving occurs for each base stepwise and discretely. This is largely different from conventional continuous motion of the sense DNA molecule 602 due to an electric field.
  • an electric field gradient driving the electrophoresis is macro and a constant force not changing with time.
  • the driving force in the present Example is micro due to thermal fluctuation and changes with time. Only a force exceeding a threshold at a certain probability and having directivity contributes to advancing the sense DNA molecule 602 . Many molecular fluctuations in the threshold do not contribute to final driving of the sense DNA molecule 602 , and the sense DNA molecule 602 stays at a potential minimum position thereof in most cases. Therefore, it is possible to detect with high accuracy a blockage current or a tunneling current depending on a base by obtaining a time average of most parts which are at potential minimum.
  • a molar polarity of the sense DNA molecule 602 passing through the nanopore 605 is limited to a 5′ ⁇ 3′ direction and a 3′ ⁇ 5′ direction.
  • the saw shape of the antisense DNA molecule 603 faces upward.
  • the antisense DNA molecule 603 is guided to the nanopore 605 by electrophoresis.
  • the temperature difference T 1 ⁇ T 2 is introduced between the antisense DNA molecule 603 and the nanopore film 604 .
  • the antisense DNA molecule 603 is in contact with the nanopore film 604 while the saw shape of the antisense DNA molecule 603 is oriented upward. Therefore, a driving force derived from the temperature to the antisense DNA molecule 603 acts upward. Therefore, as illustrated in f), the antisense DNA molecule 603 cannot invade the nanopore 605 .
  • the molar polarity of the antisense DNA molecule 603 disposed with respect to the nanopore 605 is different from the polarity of the sense DNA molecule 60 in c).
  • the polarity of a DNA molecule which can pass through the nanopore is the 5′ ⁇ 3′ direction
  • a DNA molecule having a polarity of the 3′ ⁇ 5′ direction cannot pass through the nanopore.
  • the polarity of the DNA molecule which can pass through the nanopore is the 3′ ⁇ 5 direction
  • a DNA molecule having a polarity of the 5′ ⁇ 3′ direction cannot pass through the nanopore.
  • the double stranded DNA can be unwound thermally, and only the single stranded DNA can be discriminated and selected, and can pass through the nanopore. This makes it possible to improve accuracy of analyzing abase sequence. In addition, it is possible to make the polarities of the single stranded DNA molecules have the same direction in passing through the nanopore.
  • a nanopore measurement method and a device for decoding a base sequence of a DNA molecule by driving the DNA molecule unidirectionally by introducing a temperature difference in a micro region will be described with FIG. 4 hereinafter.
  • a flow cell 211 is filled with a conductive solution including a DNA molecule 201 .
  • a nanopore film 202 separating the inside of the flow cell 211 into two portions sis and trans is present in the flow cell 211 .
  • a nanopore is formed in the nanopore film 202 .
  • the diameter of the nanopore is appropriately 5 nm or less, more preferably 2 nm or less.
  • the radius thereof is most preferably 1.2 nm.
  • the thickness of the nanopore film is appropriately 1 nm or less, more preferably 0.5 nm or less.
  • This nanopore can be formed by drilling using a focused electron beam, milling using a focused ion beam, reactive ion etching, or the like.
  • Specific examples of a material forming the nanopore film 202 include graphene. Advantages in applying graphene for decoding a DNA base sequence are the following three points.
  • Graphene has a thickness of one carbon atom, and has resolution to detect 0.34 nm which is a thickness of one nucleotide in DNA.
  • the thermal conductivity of graphene is highest in the currently known substances. A value thereof is 5000 [W/m/K]. On the other hand, the thermal conductivity of water is 0.6. There is a difference of about four orders of magnitude as the order of thermal conductivity. Therefore, these substances have ideal characteristics in introducing a temperature difference in a micro region including both of the two.
  • the DNA molecule 201 is a polymer obtained by polymerizing four kinds of nucleotides.
  • the chemical structures of the four kinds of nucleotides are very similar to each other.
  • a DNA strand is formed by regularly stacking these nucleotides spirally.
  • This DNA strand is characterized in that the DNA strand has an asymmetric form periodically because nucleotide molecules having almost the same structure are stacked.
  • This asymmetric structure is not limited to a physical three-dimensional form, but may be a profile of an electrical interaction or a chemical interaction between molecules.
  • a spring mechanism 203 is disposed at an opening of the nanopore film 202 .
  • the spring mechanism 203 presses the DNA molecule 201 to the nanopore and holds the DNA molecule 201 .
  • the DNA molecule 201 has an asymmetric shape. Therefore, the spring mechanism 203 presses a portion of the DNA molecule 201 having a small diameter.
  • Specific candidates of the spring mechanism 203 include a polymer such as nanotube or nanowire.
  • the graphene film itself also acts as the spring mechanism 203 .
  • a biological polymer such as actin filament, microtube, or a DNA single strand can be used as the spring mechanism 203 .
  • the DNA molecule 201 is electrically charged negative. Therefore, the DNA molecule 201 can be guided to the vicinity of the nanopore opening and can be brought into contact with the nanopore by applying a voltage to the solution by external electrodes 212 and 213 .
  • the spring mechanism 203 is disposed at the nanopore opening. The DNA molecule 201 cannot invade the nanopore opening and stays around the nanopore opening until the spring receives an energy s required for contracting itself from a surrounding environment.
  • the inside of the flow cell 211 is disposed in a thermostatic bath adjusted to the temperature T 1 . Therefore, the temperatures of a DNA solution in the flow cell 211 and the DNA molecule 201 in the solution are also T 1 .
  • Moving of the DNA molecule 201 to the vicinity of the nanopore opening can be detected by decrease in a blockage current. After the decrease in the blockage current is confirmed, the nanopore film 202 is cooled to the temperature T 2 using a Peltier element from the outside of the flow cell 211 by the method described in Example 2.
  • T 1 >T 2 .
  • the DNA molecule 201 performs a Brownian motion reflecting the temperature T 1 in the solution.
  • the spring mechanism 203 performs a Brownian motion reflecting the temperature T 2 of the nanopore film 202 .
  • the DNA molecule 203 stochastically acquires an energy ⁇ required for contracting the spring mechanism 203 in giving energy to or receiving energy from the heat bath. At this time, the DNA molecule 201 imparts the energy ⁇ to the spring mechanism 203 and contracts the spring mechanism 203 . As a result, the DNA molecule 201 pushes up the spring mechanism 203 , and moves downward by one base. Thereafter, the DNA molecule 201 repeats the above-described motion, and thereby passes through the nanopore. Movement by one base is generated stochastically, discontinuously, and discretely.
  • the passing speed of a DNA molecule in a nanopore can be controlled arbitrarily. More specifically, the passing speed of the DNA molecule can be reduced.
  • the DNA molecule is driven successively and discontinuously. Therefore, it is possible to detect a DNA base sequence at a higher S/N by time-averaging electric signals detecting a state of a nucleotide.
  • the DNA passes successively and discontinuously in a unit of a nucleotide, and the DNA molecule moves by one base. Thereafter, the DNA molecule stops motion at a potential minimum in one base.
  • the blockage current fluctuates, but an average thereof is a current value at the potential minimum in one base. This makes it possible to perform measurement stably by taking a time average in a sufficiently long time even when a feeble blockage current is detected.
  • Double stranded DNA is unwound thermally, and only single stranded DNA is measured selectively.
  • double stranded DNA single stranded DNAs are wound around each other complementarily and spirally. Therefore, it is necessary to examine whether a signal change obtained from a blockage current or a tunneling current is derived from the double stranded DNA or the single stranded DNA, and accuracy of the analysis is largely reduced.
  • it is possible to easily guide the single stranded DNA to a nanopore by heating a solution 209 in the flow cell 211 to a temperature (melting temperature) at which the double stranded DNA is unwound.
  • the polarity of a single stranded DNA molecule with respect to a nanopore film can be discriminated, selected, and measured. More specifically, it is possible to analyze a base sequence by selectively discriminating and selecting a polarity from a 5′ terminal to a 3′ terminal or a molar polarity opposite thereto.
  • a nanopore measurement method and a device for decoding a base sequence of a DNA molecule by driving the DNA molecule unidirectionally by introducing a temperature difference in a micro region will be described with FIG. 5 hereinafter.
  • a blockage current is used for reading the base sequence.
  • a tunneling current is used.
  • a DNA molecule 301 is electrically charged negative. Therefore, the DNA molecule 301 can be guided to the vicinity of a nanopore opening and can be brought into contact with the nanopore by applying a voltage to a solution by external electrodes 312 and 313 .
  • a spring mechanism 303 is disposed at the nanopore opening. The DNA molecule 301 cannot invade the nanopore opening and stays around the nanopore opening until the spring receives an energy ⁇ required for contracting itself from a surrounding environment.
  • a flow cell 311 is disposed in a thermostatic bath adjusted to the temperature T 1 . Therefore, the temperature of a DNA solution in the flow cell is also T 1 , and the temperature in the DNA molecule 301 in the solution is also T 1 . Moving of the DNA molecule 301 to the vicinity of the nanopore opening can be detected by decrease in the blockage current. After the decrease in the blockage current is confirmed, the nanopore film 302 is cooled to the temperature T 2 using a Peltier element from the outside of the flow cell 311 by the method described in Example 2. Here, T 1 >T 2 . By cooling, the DNA molecule 301 starts to move into the nanopore. At this time, the tunneling current is detected by microelectrodes 304 and 307 .
  • a blockage current measures an ion current passing through a nanopore. Therefore, even when a plurality of nanopores is disposed on a nanopore film, the blockage current can measure only the total of the ion currents passing through the nanopores. Therefore, parallelization in nanopore measurement is difficult.
  • each circuit for detecting a tunneling current of each of the plurality of nanopores disposed is disposed. Therefore, parallelization processing in nanopore measurement is possible.
  • An electric circuit disposed on a nanopore film is cooled to the temperature T 2 by a Peltier element outside a flow cell.
  • a noise in an electric circuit is roughly classified into a thermal noise and a shot noise.
  • the former is a random noise caused by heat of electrons.
  • the latter noise is caused because electrons flowing in an element are discrete, and cannot generate a continuous and steady flow. Both of these noises can be reduced by cooling the element in the electric circuit.
  • a tunneling current in nanopore measurement is extremely feeble. Therefore, cooling the electric circuit is effective for improving a signal noise ratio of the tunneling current.
  • the temperature difference T 2 ⁇ T 1 can be controlled arbitrarily from the outside. Therefore, a speed of the DNA molecule 301 passing through a nanopore can be also controlled arbitrarily.
  • a material of the spring mechanism 303 can be selected. Therefore, by adjusting these parameters, it is possible to set the passing speed of the DNA molecule 301 arbitrarily. It is also possible to reduce the passing speed of the DNA molecule 301 due to the external electric field only by introducing the spring mechanism 303 without introducing the temperature difference T 1 ⁇ T 2 .
  • DNA 401 does not particularly hold a spring mechanism in a nanopore film 403 .
  • the nanopore film 403 itself internally includes a spring mechanism sufficiently. Therefore, unidirectional motion of a DNA molecule due to a temperature difference can be caused without particularly introducing a spring mechanism. This brings about the following advantages. That is, in the methods and devices described in Examples 4 and 5, it is not necessary to add a particular spring mechanism, and mass production can be performed easily and inexpensively.
  • a DNA molecule 701 moves in a nanopore at 100 um/sec by applying a voltage to external electrodes 712 and 713 in a flow cell.
  • a nanopore film 702 is cooled to T 2 in advance in order to solve this problem.
  • the temperature of a solution in a flow cell 709 is T 1 , and T 1 >T 2 .
  • Example 8 a structure of a device for reversibly driving a DNA molecule in both of the upper and lower directions by introducing a temperature difference in a local micro region, controlling a driving speed, and decoding a base sequence of a DNA molecule, will be described with FIG. 8 hereinafter.
  • the temperature of a solution held by a flow cell is controlled by a thermostatic bath via an air layer. This is for maintaining the temperature of the solution in a steady state.
  • a structure of a device for inverting the temperature T 1 of a solution and the temperature T 2 of a nanopore film will be described.
  • a temperature control device illustrated in FIG. 8 can control the temperature T 1 of the solution in a flow cell 841 and the temperature T 2 of the nanopore film independently.
  • the temperature of the solution in the flow cell 841 disposed in the temperature control device is controlled to T 1 by heating a heat block 814 by a Peltier element 815 .
  • Heating control by the Peltier element 815 is performed by feedback-controlling a temperature value from a temperature measuring resistor 818 disposed in the heat block 814 . More specifically, precise temperature control is performed by PID control.
  • a region of adjusting the temperature is from 0 to 100° C.
  • an allowable temperature difference is ⁇ 0.5° C.
  • temperature stability is SD ⁇ 0.06° C. (10 minutes).
  • a thermal protector 832 is disposed in the heat block 814 . When temperature runaway of 105° C. or higher occurs, supply of a voltage to the Peltier element 815 is cut off, and heating is stopped.
  • the Peltier element 815 When the Peltier element 815 is driven, heat transfer occurs between the surface and the back surface of the Peltier element 815 due to a Seebeck effect, and a temperature difference occurs.
  • a driving efficiency of the Peltier element 815 is lowered. Therefore, a fin 816 and a fan 817 are disposed on a Peltier surface in contact with the air outside in order to reduce the temperature difference.
  • a cover 842 enhances adhesion between a flow cell 841 and the heat block 814 , and improves heat transfer.
  • the cover 842 also thermally insulates the flow cell 841 from an external environment.
  • a solution including a DNA molecule 802 is injected into the flow cell 841 disposed on the heat block 814 via a septum, and is equilibrated at the temperature T 1 .
  • a nanopore film 803 constituting a nanopore is stretched horizontally in the flow cell 841 .
  • the nanopore film 803 includes contact portions at both ends of the flow cell 841 outside, and these contact portions can be brought into contact with heat blocks 804 and 805 .
  • the heat blocks 804 and 805 can be cooled to the same temperature T 2 by driven Peltier elements 806 and 807 , respectively.
  • Temperature measuring resistors 812 and 813 which are temperature sensors are embedded in the heat blocks 804 and 805 , respectively. Temperature control is performed by PID control.
  • the heat blocks 804 and 805 are equipped with heat insulating materials 821 and 822 , respectively, in order to prevent heat transfer by direct contact between the temperature T 2 of the heat blocks 804 and 805 and the temperature T 1 in the solution.
  • Fins 808 and 809 and fans 810 and 811 are attached to the Peltier elements 806 and 807 , respectively, in order to exhaust heat generated in the Peltier elements 806 and 807 .
  • the nanopore film 803 includes one or more nanopores 801 . As described in Examples 3, the DNA 802 in the flow cell 841 is in a form of a double strand when a sample is injected, but is heated to the temperature T 1 and is unwound into single strands.
  • the DNA molecule unwound into single strands is guided to a nanopore opening by an external electrode disposed in the flow cell.
  • the temperature of each of a solution including DNA and the DNA molecule 802 is T 1 , and is controlled stably.
  • the nanopore film 803 is cooled to the temperature T 2 by the Peltier elements 806 and 807 . This makes it possible to introduce a temperature difference between the DNA molecule 802 and the nanopore opening, and to realize the circumstances described in Example 1. Therefore, the DNA molecule 802 can be driven unidirectionally.
  • the device described in the present Example can invert the temperature T 1 of a solution in the flow cell 841 and the temperature of the nanopore film 803 reversibly at a high speed.
  • the ramp rate of the solution is 5° C./sec in heating and 2.5° C./sec in cooling.
  • the ramp rate of the nanopore film is 100° C./sec in heating and 50° C./sec in cooling. This is explained by the fact that the thermal capacity of the nanopore is 1/20 or less as compared with that of a flow chip 841 .
  • the human genome having a 3G base information amount empirically requires ten times more redundancy for producing reliable data. That is, a sample having a minimum necessary information amount is required in an amount of ten times.
  • a method targeting measurement of one molecule DNA a long base length can be read advantageously, but measurement accuracy of a base sequence in one measurement is 80-85%, which is disadvantageously very low.
  • public required accuracy of a base sequence of the human genome is 99.99% or more. Therefore, it is an important object to raise measurement accuracy in the one molecule measurement method.
  • the present Example proposes a method for repeatedly reading information of a base sequence reversibly in the same molecule.
  • a single stranded DNA 901 moves successively downward in a nanopore.
  • a temperature 909 of a solution is T 1 and the temperature of the single stranded DNA 901 is similarly T 1 .
  • a nanopore film 902 is cooled, and the temperature thereof is T 2 .
  • T 1 >T 2 is cooled, and the temperature thereof.
  • T 2 ⁇ T 1 introduced between the single stranded DNA 901 and the nanopore film 902 , each base of the single stranded DNA 901 having a periodic and asymmetric structure moves downward successively and continuously.
  • the temperature T 1 in the solution is cooled to T 2 , and the temperature T 2 in the nanopore film is heated to T 1 .
  • the single stranded DNA 901 thereby moves upward as illustrated in b) to c).

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